molecular cloning 09 - | siu school of medicinebbartholomew/-lectures/molecular cloning 09.pdf ·...
TRANSCRIPT
Questions
• What is the difference between interwoundand toroidal supercoiling? – Give an example of toroidal supercoiling that
occurs inside the cell.
Supercoiling of DNA
3. DNA compaction requires special form of supercoiling
A. Interwound: supercoiling of DNA in solution
B. Toroidal- tight left handed turns, packing of DNA
both forms are interconvertible
Toroidal
Molecular Cloning:Construction of a recombinant DNA
Molecular Cloning: Cloning Vectors
• Plasmids
Grand-daddy of plasmids
Molecular Cloning: Cloning Vectors
• Plasmids– Origin of replication, determines the
number of copies per cell– Marker genes: ampicillin and tetracycline
resistance genes– Unique restriction enzyme cut sites
Creation of a polylinker or multiple cloning site
Molecular Cloning: Cloning Vectors
1. Plasmids– Origin of replication, determines the
number of copies per cell– Marker genes: ampicillin and tetracycline
resistance genes– Unique restriction enzyme cut sites
• Polylinker of MCS– Small size
• Limitation is ~15,000 bp
Questions
• How many different ways are there to construct recombinant plasmids (hint: try a Google or similar search, also check out Invitrogen)?
Invitrogen's TOPO TA Cloning System (TOPO Cloning)
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications
Figure 2. Gateway® technology facilitates cloning of genes, into and back out of, multiple vectors via site-specific recombination.Once a gene is cloned into an Entry clone you can then move the DNA fragment into one or more destination vectors simultaneously.
Entry of DNA into cells
• Chemical Transformation– Treat cells with calcium chloride– Heat shock
• Electroporation– High voltage transiently makes bacterial
membrane permeable
Special examples of different plasmids
• Expression vectors
What are the considerations in vector construction that need to be taken into account with over-
expression of a protein?
Questions
• Why are protein expression vectors useful?• What kind of cells are proteins generally
over-expressed in and why?• What are the considerations in vector
construction that need to be taken into account with over-expression of a protein?
Special examples of different plasmids
A. Expression vectors– Strong promoter for transcription– Ribosome binding site– Transcription terminator– Some way of controlling gene – making it
inducible
Fusion proteins
Tag expressed protein with another protein or a short peptide
Type of Tags
• Fluorescent proteins– One example is the green fluorescent
protein or GFP
Type of Tags
• Fluorescent proteins– Examples is the green fluoresecent protein
or GFP• Epitope tag
– Short peptide sequence which has an antibody that recognizes it specifically
Type of Tags
• Fluorescent proteins– Examples is the green fluoresecent protein or
GFP• Epitope tag
– Short peptide sequence which has an antibody that recognizes it specifically
• Metal chelator– Can bind to Ni or Co chelated and immobolized
Special examples of different plasmids
B. Shuttle vectors– Contains origins of replication from two different
organisms– Can be replicated in both – Often used to shuttle plasmids from bacteria to
yeastC. Cosmids
- Plasmids containing at least one cos (cohesive-end site) of lambda phage
- Up to 44 kbps
Molecular Cloning: Cloning Vectors
2. Bacteriophages: example is lambda– 1/3 of genome (48.5 Kb) is non essential– DNA is packaged into phage particles– Can only fit 40 – 53 Kb of DNA – Have an in vitro packaging system– Highly efficient at transforming bacteria – Can clone up to 23 Kb of DNA
Molecular Cloning: Cloning Vectors
3. Bacterial Artificial Chromosomes (BAC)– 100 to 300 Kb in size– Have selectable markers– Stable origin of replication– Size of inserts is ~100 KB– Uses electroporation
Molecular Cloning: Cloning Vectors
4. Yeast Artificial Chromosomes
Molecular Cloning: Cloning Vectors
4. Yeast Artificial Chromosomes (YAC)– ARS or origin of replication– Selectable markers– CEN or centromere sequence for proper
segregation– Telomere sequences– Suitable for very large DNAs
A DNA library
• Collection of DNA clones• Source for gene discovery• Largest example is genomic library• Other subsets would be such things as:
– cDNA libraries• Can scan these libraries by DNA
hybridization
• Why are cDNA libraries still so important in the era of genomics?
• What information can be derived from cDNA that are not obtained generally by DNA microarrays?
Full-length cDNA clones and the sequence information derived thereof are the entry to:
• Proteomics : Only full-length cDNAs enable the synthesis, use, and analysis of proteins.
• Gene Regulation : Only full-length cDNAs allow for promoter identification, and as such give you access to the regulatory information that drives gene expression.
• RNAi : The more information you have the greater your chances to prepare a specific RNAi probe.
• Splicing : Only full-length cDNA information can tell you about the true exon composition of transcripts.
• Genomics : Full-length cDNAs are needed for gene discovery and correct mapping of genomic locations.
• SNP Analysis : Only a complete and correct annotation of a gene enables functional characterization of SNPs.
Next time on to protein methods
Change in the syllabus – will not be doing protein structure in this section
Questions
• Why would you need to purify protein?• What properties of proteins can be used to
separate and purify them from each other?• What is column chromatography and how
does it apply to protein purification?• What causes proteins to denature?